DNA coding for a protein which imparts L-homoserine resistance to Escherichia coli bacterium, and a method for producing L-amino acids

ABSTRACT

A bacterium which has an ability to produce an amino acid and in which a novel rhtB gene encodes a protein having an activity of making a bacterium having the protein L-homoserine-resistant is enhanced, is cultivated in a culture medium to produce and cause accumulation of the amino acid in the medium, and the amino acid is recovered from the medium.

This application claims benefit as a continuation under 35 U.S.C. §120to U.S. patent application Ser. No. 09/847,392, which is a divisional of09/396,357, now U.S. Pat. No. 6,303,348.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method for producing an amino acid,and especially a method for producing L-homoserine, L-alanine,L-isoleucine, L-valine, or L-threonine using a bacterium belonging tothe genus Escherichia.

2. Background Art

The present inventors obtained, with respect to E. coli K-12, a mutanthaving a mutation thrR (hereinafter, “rhtA23”) that results in highconcentrations of threonine (>40 mg/ml) or homoserine (>5 mg/ml) in aminimal medium (Astaurova, O. B. et al., Appl. Bioch. and Microbiol.,21, 611-616 (1985)). On the basis of the rhtA23 mutation, an improvedthreonine-producing strain (SU patent No. 974817), and homoserine- andglutamic acid-producing strains (Astaurova et al., Appl. Boch. AndMicrobiol., 27, 556-561 (1991)) were obtained.

Furthermore, the present inventors have reported that the rhtA geneexists at 18 min on the E. coli chromosome, and is identical to the ORF1between pexB and ompX genes. DNA expressing a protein encoded by theORF1 has been designated the rhtA (rht: resistance to homoserine andthreonine) gene. The rhtA gene includes a 5′-noncoding region, whichincludes a SD sequence, an ORF1, and a terminator. Also, the presentinventors have found that a wild-type rhtA gene imparts resistance tothreonine and homoserine if cloned in a multi-copy state, and thatenhancement of expression of the rhtA gene improves amino acidproductivity of a bacterium belonging to the genus Escherichia which hasan ability to produce L-lysine, L-valine or L-threonine (ABSTRACTS of17th International Congress of Biochemistry and Molecular Biology inconjugation with 1997 Annual Meeting of the American Society forBiochemistry and Molecular Biology, San Francisco, Calif. Aug. 24-29,1997, abstract No. 457).

The present inventors have found during the cloning of the rhtA genethat at least two distinct genes which impart homoserine resistance in amulti-copy state exist in E. coli. One is the rhtA gene, and the otherhas not been previously reported.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel gene whichimparts resistance to homoserine, and a method for producing an aminoacid, especially, L-homoserine, L-alanine, L-isoleucine, L-valine, andL-threonine with a high yield.

The inventors have found that a region at 86 min on the E. colichromosome, when cloned into a multi-copy vector, is able to impartresistance to L-homoserine to E. coli, and that when the region isamplified, the amino acid productivity of the E. coli is improved,similar to the rhtA gene. On the basis of these findings, the presentinvention has been completed.

It is an object of the present invention to provide a DNA coding for aprotein selected from the group consisting of:

-   -   (A) a protein comprising an amino acid sequence shown in SEQ ID        NO: 2; and    -   (B) a protein comprising an amino acid sequence which includes        deletion, substitution, insertion, or addition of one or several        amino acids in the amino acid sequence shown in SEQ ID NO: 2,        and wherein said protein has an activity of making a bacterium        having the protein L-homoserine-resistant,

It is an object of the present invention to provide the DNA as describedabove which is a DNA selected from the group consisting of:

-   -   (a) a DNA comprising a nucleotide sequence corresponding to        numbers 557 to 1171 shown in SEQ ID NO: 1; and    -   (b) a DNA which is able to hybridize with the nucleotide        sequence of numbers 557 to 1171 shown in SEQ ID NO: 1 under        stringent conditions, and wherein said DNA codes for a protein        having the activity of making the bacterium having the protein        L-homoserine-resistant.

It is an object of the present invention to provide a bacteriumbelonging to the genus Escherichia, wherein L-homoserine resistance ofthe bacterium is enhanced by amplifying a copy number of theabove-described DNA in the bacterium.

It is a further object of the present invention to provide the bacteriumas described above wherein the above-described DNA is carried on amulticopy vector in the bacterium,

It is an object of the present invention to provide the bacterium asdescribed above wherein the above-described DNA is carried on atransposon in the bacterium.

It is an object of the present invention to provide a method forproducing an amino acid, comprising cultivating the bacterium asdescribed above which has an ability to produce the amino acid, in aculture medium to produce and cause accumulatation of the amino acid inthe medium, and recovering the amino acid from the medium.

It is an object of the present invention to provide the method asdescribed above, wherein the amino acid is at least one selected fromthe group consisting of L-homoserine, L-alanine, L-isoleucine, L-valine,and L-threonine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cloning, identification, and inactivation of the rhtBgene.

FIG. 2 shows the amino acid sequence of the RhtB protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The DNA of the present invention may be referred to as the “rhtB gene.”A protein encoded by the rhtB gene may be referred to as the “RhtBprotein.” An activity of the RhtB protein which imparts resistance toL-homoserine to a bacterium, i.e. an activity of making a bacteriumhaving the RhtB protein L-homoserine-resistant, may be referred to asthe “Rh activity.” A structural gene encoding the RhtB protein withinthe rhtB gene may be referred to as the “rhtB structural gene”. Thephrase “enhancing the Rh activity” means imparting resistance tohomoserine to a bacterium or enhancing the resistance by increasing thenumber of RhtB protein molecules, increasing a specific activity of theRhtB protein, or desensitizing negative regulation of the expression orthe activity of the RhtB protein, or the like. The phrase “DNA codingfor a protein” means a double-stranded DNA in which one strand codes forthe protein. “L-homoserine resistance” means that a bacterium is able togrow on a minimal medium containing L-homoserine at a concentration atwhich a wild-type strain thereof cannot grow, usually 10 mg/ml. Abacterium having an “ability to produce an amino acid” means that thebacterium produces and is able to cause accumulation of larger amountsof an amino acid in a medium than a wild-type strain thereof.

According to the present invention, resistance to a high concentrationof homoserine can be imparted to a bacterium belonging to the genusEscherichia. A bacterium belonging to the genus Escherichia, which hasan increased resistance to homoserine and an ability to causeaccumulation of an amino acid, especially, L-homoserine, L-alanine,L-isoleucine, L-valine, or L-threonine in a medium with a high yield isdisclosed.

The present invention will be explained in detail below.

<1> DNA of the Present Invention

The DNA of the present invention encodes a protein which has Rh activityand an amino acid sequence of SEQ ID NO: 2. Specifically, the DNA of thepresent invention may be exemplified by a DNA comprising a nucleotidesequence of the numbers 557 to 1171 in SEQ ID NO: 1.

The DNA of the present invention includes a DNA fragment encoding theRhtB protein which imparts resistance to homoserine to the bacteriumEscherichia coli. The DNA of the present invention includes a DNAfragment which includes the regulatory elements of the rhtB gene and thestructural part of rhtB gene, and which has the nucleotide sequenceshown in SEQ ID NO: 1.

The nucleotide sequence shown in SEQ ID NO: 1 corresponds to a part of asequence complementary to the sequence of GenBank accession numberM87049. SEQ ID NO: 1 includes f138 (nucleotide numbers 61959-61543 ofGenBank accession number M87049) which is a known ORF (open readingframe) located at 86 min on the E. coli chromosome, and 5′-flanking and3′-flanking regions thereof, but the function of f138 is unknown. Thef138, which contains only 160 nucleotides in the 5′-flanking region,cannot impart resistance to homoserine. No termination codon is presentbetween the 62160 and 61959 of M87049 (upstream to the ORF f138). Hence,the coding region is 201 bp longer. Thus, the RhtB protein and the rhtBgene are novel.

The rhtB gene may be obtained, for example, by infecting Mucts lysogenicstrain of E. coli using a lysate of a lysogenic strain of E. coli suchas K12 or W3110 according to the method using mini-Mu d5005 phagemid(Groisman, E. A., et al., J. Bacteriol., 168, 357-364 (1986)), andisolating plasmid DNAs from colonies grown on a minimal mediumcontaining kanamycin (40 μg/ml) and L-homoserine (10 mg/ml). Asillustrated in the Example described below, the rhtB gene was mapped at86 min on the chromosome of E. coli. Therefore, the DNA fragmentincluding the rhtB gene may be obtained from the chromosome of E. coliby colony hybridization or PCR (polymerase chain reaction, refer toWhite, T. J. et al, Trends Genet. 5, 185(1989)) using anoligonucleotide(s) which has a sequence corresponding to the region nearthe portion at 86 min on the chromosome of E. coli. Alternatively, theoligonucleotide may be designed according to the nucleotide sequenceshown in SEQ ID NO: 1. By using oligonucleotides having nucleotidesequences corresponding to an upstream region from nucleotide number 557and a downstream region from nucleotide number 1171 in SEQ ID NO: 1 asthe primers for PCR, the entire coding region can be amplified.

Synthesis of the oligonucleotides can be performed by an ordinary methodsuch as the phosphoamidite method (see Tetrahedron Letters, 22, 1859(1981)) by using a commercially available DNA synthesizer (for example,DNA Synthesizer Model 380B produced by Applied Biosystems). Furthermore,PCR can be performed using a commercially available PCR apparatus (forexample, DNA Thermal Cycler Model PJ2000 produced by Takara Shuzo Co.,Ltd.), using Taq DNA polymerase (supplied by Takara Shuzo Co., Ltd.) inaccordance with a method designated by the supplier.

The DNA coding for the RhtB protein of the present invention may codefor a RhtB protein which includes deletion, substitution, insertion, oraddition of one or several amino acids at one or a plurality ofpositions, provided the Rh activity of RhtB protein encoded thereby isnot disrupted. The DNA, which codes for substantially the same proteinas the RhtB protein described above, may be obtained, for example, bymodifying the nucleotide sequence, for example, by means of thesite-directed mutagenesis method so that one or more amino acid residuesat a specified site is deleted, substituted, inserted, or added. DNAmodified as described above may be obtained by conventionally knownmutation treatments. These treatments includes treating DNA coding forthe RhtB protein in vitro, for example, with hydroxylamine, and treatinga microorganism, for example, a bacterium belonging to the genusEscherichia harboring a DNA coding for the RhtB protein with ultravioletirradiation or a mutating agent such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid, usuallyused for the mutation treatment.

The DNA which codes for substantially the same protein as the RhtBprotein can be obtained by expressing a DNA which has been subjected toin vitro mutation treatment as described above in a multi-copy vector inan appropriate cell, investigating the resistance to homoserine, andselecting the DNA which imparts an increase in the resistance. Also, itis generally known that an amino acid sequence of a protein and anucleotide sequence coding for it may be slightly different betweenspecies, strains, mutants, or variants, and therefore the DNA, whichcodes for substantially the same protein, can be obtained fromL-homoserine-resistant species, strains, mutants, and variants belongingto the genus Escherichia. Specifically, the DNA, which codes forsubstantially the same protein as the RhtB protein, can be obtained byisolating a DNA which hybridizes with DNA having, for example, anucleotide sequence of numbers 557 to 1171 shown in SEQ ID NO: 1 understringent conditions, and which codes for a protein having Rh activity,from a bacterium belonging to the genus Escherichia which has beensubjected to a mutation treatment, or a spontaneous mutant or variant ofa bacterium belonging to the genus Escherichia. The term “stringentconditions” referred to herein are conditions under which a so-calledspecific hybrid is formed, and a non-specific hybrid is not formed. Itis difficult to clearly express this condition by using any numericalvalue. However, for example, the stringent conditions include thoseunder which DNAs having high homology, for example, DNAs having homologyof not less than 70% to each other are able to hybridize, and DNAshaving homology lower than the above to each other are not able tohybridize.

<2> Bacterium Belonging to the Genus Escherichia of the PresentInvention

The bacterium belonging the genus Escherichia of the present inventionis a bacterium belonging to the genus Escherichia having enhanced Rhactivity. A bacterium belonging to the genus Escherichia is exemplifiedby Escherichia coli. The Rh activity can be enhanced by, for example,amplifying the copy number of the rhtB structural gene in a cell, ortransforming a bacterium belonging to the genus Escherichia with arecombinant DNA which includes the rhtB structural gene encoding theRhtB protein ligated to a promoter sequence which functions efficientlyin a bacterium belonging to the genus Escherichia. The Rh activity canbe also enhanced by substituting the promoter sequence of the rhtB geneon a chromosome with a promoter sequence which functions efficiently ina bacterium belonging to the genus Escherichia.

The amplification of the copy number of the rhtB structural gene in acell can be performed by introducing a multi-copy vector which carriesthe rhtB structural gene into a bacterium belonging to the genusEscherichia. Specifically, the copy number can be increased byintroduction of a plasmid, a phage, or a transposon (Berg, D. E. andBerg, C. M., Bio/Technol., 1, 417 (1983)) which carries the rhtBstructural gene into a bacterium belonging to the genus Escherichia.

Examples of a multicopy vector include plasmid vectors such as pBR322,pMW118, pUC19, or the like, and phage vectors such as λ1059, λBF101, M13mp9, or the like. The transposon is exemplified by Mu, Tn10, Tn5, or thelike.

The introduction of a DNA into a bacterium belonging to the genusEscherichia can be performed, for example, by the method of D. M.Morrison (Methods in Enzymology 68, 326 (1979)), or a method in whichrecipient bacterial cells are treated with calcium chloride to increasethe permeability of DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159(1970)), and the like.

If the Rh activity is enhanced in an amino acid-producing bacteriumbelonging to the genus Escherichia as described above, the amount ofamino acid produced can be increased. As the bacterium belonging to thegenus Escherichia which is to have the Rh. activity enhanced, strainswhich have abilities to produce desired amino acids are used.Alternatively, an ability to produce an amino acid may be imparted to abacterium in which the Rh activity is enhanced. Examples of aminoacid-producing bacteria belonging to the genus Escherichia are describedbelow.

L-Threonine-Producing Bacteria

The L-threonine-producing bacteria belonging to the genus Escherichiamay be exemplified by strain MG442 (Guayatiner et al., Genetika (inRussian), 14, 947-956 (1978)).

(2) L-Homoserine-Producing Bacteria

The L-homoserine-producing bacteria belonging to the genus Escherichiamay be exemplified by strain NZ10 (thrB). This strain was derived fromthe known strain C600 (thrB, leuB) (Appleyard R. K., Genetics, 39,440-452 (1954)) and is Leu⁺ revertant.

On the basis of the rhtB DNA fragment, new amino acid-producing strainsE. coli NZ10/pAL4, pRhtB; E. coli MG422/pVIC40, pRhtB; and E. coliMG442/pRhtB were obtained which are useful for the production of aminoacids by fermentation.

The new strains have been deposited in accordance with the BudapestTreaty in the Russian National Collection of Industrial Microorganisms(VKPM) on Oct. 6, 1998. The strain E. coli NZ10/pAL4, pRhtB wasdeposited and given accession number VKPM B-7658; the strain E. coliMG442/pRhtB was deposited and given accession number of VKPM B-7659; andthe strain E. coli MG442/pVIC40,pRhtB was deposited and given accessionnumber of VKPM B-7660.

The strain E. coli NZ10/pAL4, pRhtB (VKPM B-7658) exhibits the followingculture, morphological, and biochemical features.

Cytomorphology: Gram-negative weakly-motile rods having rounded ends.Longitudinal size: 1.5 to 2 μm.

Culture Features:

Beef-extract agar—After 24-hour growth at 37° C., produces round whitishsemitransparent colonies 1.5 to 3 mm in diameter, featuring a smoothsurface, regular or slightly wavy edges, the center is slightly raised,homogeneous structure, pastelike consistency, readily emulsifiable.

Luria's agar—After a 24-hour growth at 37° C., develops whitishsemitranslucent colonies 1.5 to 2.5 mm in diameter having a smoothsurface, homogeneous structure, pastelike consistency, readilyemulsifiable.

Minimal agar-doped medium M9—After 40 to 48 hours of growth at 37° C.,forms colonies 0.5 to 1.5 mm in diameter, which are greyish-white incolor, semitransparent, slightly convex, with a lustrous surface.

Growth in a beef-extract broth—After 24-hour growth at 37° C., exhibitsstrong uniform cloudiness, has a characteristic odor.

Physiological and Biochemical Features:

Grows upon thrust inoculation in a beef-extract agar. Exhibits goodgrowth throughout the inoculated area. The microorganism proves to be afacultative anaerobe.

It does not liquefy gelatin.

Features good growth on milk, accompanied by milk coagulation.

Does not produce indole.

Temperature conditions—Grows on beef-extract broth at 20-42° C., anoptimum temperature being within 33-37° C.

pH value of culture medium—Grows on liquid media having the pH valuefrom 6 to 8, an optimum value being 7.2.

Carbon sources—Exhibits good growth on glucose, fructose, lactose,mannose, galactose, xylose, glycerol, and mannitol to produce an acidand gas.

Nitrogen sources—Assimilates nitrogen in the form of ammonium, nitricacid salts, as well as from some organic compounds.

Resistant to ampicillin, kanamycin and L-homoserine.

L-Threonine is used as a growth factor.

Content of plasmids—The cells contain the multi-copy hybrid plasmid pAL4which ensures resistance to ampicillin and carries the thrA gene fromthe threonine operon, which codes for aspartate kinase-homoserinedehydrogenase I and is responsible for increased homoserinebiosynthesis. Besides, the cells contain a multi-copy hybrid plasmidpRhtB which ensures resistance to kanamycin and carries the rhtB genewhich confers resistance to homoserine (10 mg/l).

The strain E. coli MG442/pRhtB (VKPM B-7659) has the same culture,morphological, and biochemical features as the strain NZ10/pAL4, pRhtB,except that L-isoleucine is used as a growth factor instead ofL-threonine. However, the strain can grow slowly without isoleucine.Besides, the cells of the strain contain only one multi-copy hybridplasmid pRhtB which ensures resistance to kanamycin and carryies therhtB gene which confers resistance to homoserine (10 mg/l).

The strain E. coli MG442/pVIC40,pRhtB (VKPM B-7660) has the sameculture, morphological, and biochemical features as the strainNZ10/pAL4, pRhtB, except for L-isoleucine is used as a growth factorinstead of L-threonine. However, the strain can grow slowly withoutisoleucine. The cells of the strain contain the multi-copy hybridplasmid pVIC40 which ensures resistance to streptomycin and carryies thegenes of the threonine operon. Besides, they contain multi-copy hybridplasmid pRhtB which ensures resistance to kanamycin and carryies therhtB gene which confers resistance to homoserine (10 mg/l).

<3> Method for Producing an Amino Acid

An amino acid can be efficiently produced by cultivating the bacteriumhaving enhanced Rh activity by amplifying a copy number of the rhtB geneas described above, and which has an ability to produce the amino acid,in a culture medium, producing and causing accumulation of the aminoacid in the medium, and recovering the amino acid from the medium. Theamino acid is exemplified preferably by L-homoserine, L-alanine,L-isoleucine, L-valine, and L-threonine.

In the method of present invention, the cultivation of the bacteriumbelonging to the genus Escherichia, and the collection and purificationof an amino acid from the liquid medium may be performed in a mannersimilar to conventional methods for producing an amino acid byfermentation using a bacterium. The cultivation medium may be eithersynthetic or natural, so long as the medium includes a carbon andnitrogen source and minerals and, if necessary, nutrients which thechosen bacterium requires for growth in appropriate amounts. The carbonsource may include various carbohydrates such as glucose and sucrose,and various organic acids. Depending on the assimilatory ability of thechosen bacterium, alcohol including ethanol and glycerol may be used. Asthe nitrogen source, ammonia, various ammonium salts such as ammoniumsulfate, other nitrogen compounds such as amines, a natural nitrogensource such as peptone, soybean hydrolyte, and digested fermentativemicrobe can be used. As minerals, monopotassium phosphate, magnesiumsulfate, sodium chloride, ferrous sulfate, manganese sulfate, andcalcium carbonate can be used.

The cultivation is preferably performed under aerobic conditions such asshaking, aeration, and stirring. The temperature of the culture isusually 20 to 40° C., preferably 30 to 38° C. The pH of the culture isusually between 5 and 9, preferably between 6.5 and 7.2. The pH of theculture can be adjusted with ammonia, calcium carbonate, various acids,various bases, and buffers. Usually, a 1 to 3-day cultivation leads tothe accumulation of the target amino acid in the medium.

Recovering the amino acid after cultivation can be performed by removingsolids such as cells from the medium by centrifugation or membranefiltration, and then collecting and purifying the target amino acid byion exchange, concentration, and crystalline fraction methods, and thelike.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting Examples. In the Examples, anamino acid is of L-configuration unless otherwise noted.

Example 1 Obtaining the rhtB DNA Fragment

Cloning the rhtB Gene into Mini-Mu Phagemid

The wild-type rhtB gene was cloned in vivo using mini-Mu d5005 phagemid(Groisman, E. A., et al., J. Bacteriol., 168, 357-364 (1986)). MuCts62lysogen of the strain MG442 was used as a donor. Freshly preparedlysates were used to infect a Mucts lysogenic derivative of a strainVKPM B-513 (Hfr K10 metB). The cells were plated on M9 glucose minimalmedium with methionine (50 μg/ml), kanamycin (40 μg/ml) and homoserine(10 mg/ml). Colonies which appeared after 48 hr were picked andisolated. Plasmid DNA was isolated and used to transform the strain VKPMB-513 by standard techniques. Transformants were selected on L-brothagar plates with kanamycin as above. Plasmid DNA was isolated from thosewhich were resistant to homoserine, and the inserted fragments wereanalyzed by restriction mapping. It appeared that two types of insertsbelonging to different chromosome regions had been cloned from thedonor. Thus, at least two different genes are present in multi-copy formand impart resistance to homoserine exist in E. coli. One of the twotypes of inserts is the rhtA gene which has already reported (ABSTRACTSof 17th International Congress of Biochemistry and Molecular Biology inconjugation with 1997 Annual Meeting of the American Society forBiochemistry and Molecular Biology, San Francisco, Calif. Aug. 24-29,1997). The other type of insert is a fragment having a minimum lengthwhich imparts resistance to homoserine of 0.8 kb (FIG. 1).

(2) Identification of rhtB Gene

The insert fragment was sequenced by the dideoxy chain terminationmethod of Sanger. Both DNA strands were sequenced in their entirety andall junctions were overlapped. The sequencing showed that the insertfragment included f138 (nucleotide numbers 61543 to 61959 of GenBankaccession number M87049) which was a known ORF (open reading frame)present at 86 min of E. coli chromosome and 201 bp of an upstream regionthereof (downstream region in the sequence of M87049), but the functionis unknown. The f138 having only 160 nucleotides in the 5′-flankingregion was not able to impart resistance to homoserine. No terminationcodon is present upstream the ORF f138 between 62160 and 61959nucleotides of M87049. Furthermore, one ATG following a predictedribosome binding site is present in the sequence. The larger ORF(nucleotide numbers 62160 to 61546) is designated as the rhtB gene. TheRhtB protein deduced from the gene is highly hydrophobic and contains 5possible transmembrane segments.

Example 2 Production of Homoserine-Producing Strain

Strain NZ10 of E. coli was transformed with the plasmid pAL4 to obtainthe strain NZ10/pAL4. This plasmid was constructed by inserting a thrAgene, which encodes aspartokinase-homoserine dehydrogenase I, into apBR322 vector. The strain NZ10 is a leuB⁺-reverted mutant (thrB)obtained from the E. coli strain C600 (thrB, leuB) (Appleyard, Genetics,39, 440-452 (1954)).

The rhtB gene was inserted into plasmid pUK21, which is a known plasmidpUC19 having a kanamycin resistance gene substituted for an ampicillinresistance gene (Vieira, J. and Messing, J., Gene, 100, 189-194 (1991)),to obtain pRhtB.

The strain NZ10/pAL4 was transformed with pUK21 or pRhtB to obtainstrains NZ 10/pAL4, pUK21, and NZ 10/pAL4, pRhtB.

The thus obtained transformants were each cultivated at 37° C. for 18hours in a nutrient broth with 50 mg/l kanamycin and 100 mg/lampicillin, and 0.3 ml of the obtained culture was inoculated into 3 mlof a fermentation medium having the following composition and containing50 mg/l kanamycin and 100 mg/l ampicillin, in a 20×200 mm test tube, andcultivated at 37° C. for 46 hours with a rotary shaker. Aftercultivation, the amount of homoserine which accumulates in the medium,and the absorbance at 560 nm of the medium were determined by knownmethods.

Fermentation medium composition (g/L) Glucose 80 (NH₄)₂SO₄ 22 K₂HPO₄ 2NaCl 0.8 MgSO₄ 7H₂O 0.8 FeSO₄ 7H₂O 0.02 MnSO₄ 5H₂O 0.02 Thiaminehydrochloride 0.0002 Yeast Extract 1.0 CaCO₃ 30 (CaCO₃ was separatelysterilized)

The results are shown in Table 1. The strain NZ10/pAL4, pRhtB was ableto cause accumulation of a larger amount of homoserine than the strainsNZ 10/pAL4 and NZ 10/pAL4, pUK21, which do not have an enhanced rhtBgene. TABLE 1 Accumulated amount of Strain OD₅₆₀ homoserine (g/L)NZ10/pAL4 16.4 3.1 NZ10/pAL4, pUK21 14.3 3.3 NZ10/pAL4, pRhtB 15.6 6.4

Example 3 Production of Alanine, Valine, and Isoleucine Using aRhtB-Transformed Strain

E. coli strain MG442 is a known strain (Gusyatiner, et al., 1978,Genetika (in Russian), 14:947-956).

The strain MG442 was transformed with the plasmids pUK21 and pRhtB toobtain strains MG442/pUK21 and MG442/pRhtB.

The thus obtained transformants were each cultivated at 37° C. for 18hours in a nutrient broth with 50 mg/l kanamycin, and 0.3 ml of theobtained culture was inoculated into 3 ml of the fermentation mediumdescribed in Example 3, which contains 50 mg/l kanamycin, in a 20×200 mmtest tube, and cultivated at 37° C. for 40 hours with a rotary shaker.After cultivation, accumulated amounts of alanine, valine and isoleucinein the medium and an absorbance at 560 nm of the medium were determinedby known methods.

The results are shown in Table 2. The strain MG442/pRhtB was able tocause accumulation of larger amounts of alanine, valine, and isoleucinethan the strain MG442/pUK21, which does not have an enhanced rhtB gene.TABLE 2 Accumulated amount (g/L) Strain OD₅₆₀ Alanine Valine IsoleucineMG442/pUK21 13.4 0.2 0.2 0.3 MG442/pRhtB 13.7 0.7 0.5 0.5

Example 4 Production of Threonine-Producing Strain

The strain MG442 (Example 3) was transformed with the known plasmidpVIC40 (U.S. Pat. No. 5,175,107 (1992)) by an ordinary transformationmethod. Transformants were selected on LB agar plates containing 0.1mg/ml streptomycin. Thus a novel strain MG422/pVIC40 was obtained.

The strain MG442/pVIC40 was transformed with pUK21 or pRhtB to obtainstrains MG442/pVIC40, pUK21 and MG442/pVIC40, pRhtB.

The thus obtained transformants were each cultivated at 37° C. for 18hours in a nutrient broth with 50 mg/l kanamycin and 100 mg/lstreptomycin, and 0.3 ml of the obtained culture was inoculated into 3ml of the fermentation medium described in Example 3, which contains 50mg/l kanamycin and 100 mg/l streptomycin, in a 20×200 mm test tube, andcultivated at 37° C. for 46 hours with a rotary shaker. Aftercultivation, the accumulated amount of threonine in the medium, and anabsorbance at 560 nm of the medium were determined by known methods.

The results are shown in Table 3. The strain MG442/pVIC40, pRhtB wasable to cause accumulation of larger amounts of threonine than thestrains MG442/pVIC40 and MG442/pVIC40,pUK21, which do not have anenhanced rhtB gene. TABLE 3 Accumulated amount of Strain OD₅₆₀ threonine(g/L) MG442/pVIC40 17   13.6 MG442/pVIC40, pUK21 16.3 12.9 MG442/pVIC40,pRhtB 15.2 16.3

Example 5 Effect of rhtB Gene Inactivation and Amplification onBacterium E. Coli Resistance to Some Amino Acids and Amino AcidAnalogues

To inactivate the chromosomal rhtB gene, the plasmid pNPZ46 wasconstructed (FIG. 1) on the basis of pUK21 vector. It harbors a DNAfragment from 86 min of E. coli chromosome, with the rhtB gene and5′-flanking and 3′-flanking regions thereof. Then, the ClaI-Eco47IIIfragment of the pNPZ46 plasmid rhtB gene was substituted for anAsuII-BsrBI fragment containing a cat (Cm^(R)) gene from the pACYC184plasmid (Chang and Cohen, J. Bacteriol., 134, 1141-1156, 1978), givingthe pNPZ47 plasmid (FIG. 1). To introduce the obtained insertionallyinactivated rhtB gene into the chromosome of the E. coli strain N99 (thestreptomycin-resistant derivative of the known strain W3350 (Campbell,Virology, 14, 22-33, 1961)), the method of Parker and Marinus was used(Parker, B. and Marinus, M. G., Gene, 73, 531-535, 1988). Thesubstitution of the wild-type allele for the inactivated one wasaccomplished by phage P1 transduction and Southern hybridization(Southern, E. M., J. Mol. Biol., 98, 503-517, 1975).

Then the susceptibility of the thus obtained E. coli strain N99rhtB::cat, of the initial strain N99 (rhtB⁻), and of its derivativetransformed with pRhtB plasmid, N99/pRhtB, to some amino acids and aminoacid analogues was tested. Overnight cultures of the strains grown in M9minimal medium at 37° C. with a rotary shaker (10⁹ cfu/ml) were diluted1:100 and grown for 5 hours under the same conditions. Then the logphase cultures thus obtained were diluted and about 10⁴ live cells wereapplied to well-dried test plates with M9 agar containing doublingincrements of amino acids or analogues. The minimum inhibitoryconcentration (MIC) of these compounds was examined after a 40-46 hcultivation. The results are shown in Table 4. TABLE 4 MIC (μg/ml)Substrate N99 (rhtB⁺) N99/pRhtB N99 rhtB::cat 1. L-homoserine 250 30000125 2. L-threonine 30000 50000 30000 3. L-serine 5000 10000 5000 4.L-valine 0.5 1 0.5 5. AHVA 50 2000 25 6. AEC 10 25 10 7.4-aza-DL-leucine 40 100 40

It follows from Table 4 that multiple copies of rhtB imparted to cellsincreased resistance to threonine, serine, valine,α-amino-β-hydroxyvaleric-acid (AHVA), S-(2-aminoethyl)-L-cysteine (AEC),and 4-aza-DL-leucine. The inactivation of the rhtB gene, on thecontrary, increased the cells' sensitivity to homoserine and AHVA. Theseresults in conjunction with the data on homology of the RhtB protein tothe LysE lysine efflux transporter of Corynebacterium glutamicum (Vrljicet al., Mol. Microbiol., 22, 815-826, 1996) indicate the presence offunctional analogues for the rhtB gene product. The presumed effluxtransporter, RhtB, has specificity to several substrates (amino acids),or may show non-specific effects as a result of amplification.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. The aforementioned documents,as well as the foreign priority document, RU98118425, and the parentapplication Ser. Nos. 09/847,392 and 09/396,357 which is now U.S. Pat.No. 6,303,348, are incorporated by reference herein in their entirety.

1. A method for producing an amino acid selected from the groupconsisting of L-threonine, L-homoserine, L-alanine, L-isoleucine, andL-valine comprising: A) cultivating a bacterium which has an ability toproduce an amino acid in a medium, and b) recovering said amino acidfrom the medium, wherein said bacterium belongs to the genusEscherichia, and wherein L-homoserine resistance of said bacterium isenhanced by amplifying a copy number of a DNA in said bacterium, whereinsaid DNA is able to hybridize under stringent conditions to nucleotides557 to 1171 of SEQ ID NO: 1, wherein said DNA is not less than 70%homologous to nucleotides 557 to 1171 of SEQ ID NO: 1, and wherein saidDNA encodes a protein which has an activity of imparting L-homoserineresistance to a bacterium having the protein.
 2. The method according toclaim 1, wherein said amino acid is at least one selected from the groupconsisting of L-alanine, L-isoleucine, and L-valine.
 3. The methodaccording to claim 1, wherein said DNA is carried on a multi-copyvector.
 4. The method according to claim 1, wherein said DNA is carriedon a transposon.
 5. A method for producing an amino acid selected fromthe group consisting of L-threonine, L-homoserine, L-alanine,L-isoleucine, and L-valine comprising: A) cultivating a Escherichiabacterium which has been transformed with a mutant DNA in a medium,wherein said Escherichia bacterium has an ability to produce the aminoacid, and B) recovering the amino acid from the medium, wherein saidmutant DNA is obtainable by mutating a DNA comprising nucleotides 557 to1171 of SEQ ID NO: 1 and selecting a mutant DNA which, when transferredinto a bacterium increases the homoserine resistance of the bacterium ascompared to the bacterium prior to receiving said mutant DNA, andwherein said mutant DNA is not less than 70% homologous to nucleotides557 to 1171 of SEQ ID NO:1.
 6. The method according to claim 5, whereinsaid amino acid is at least one selected from the group consisting ofL-alanine, L-isoleucine, and L-valine.
 7. The method according to claim5, wherein said DNA is carried on a multicopy vector.
 8. The methodaccording to claim 5, wherein said DNA is carried on a transposon. 9.The method according to claim 5, wherein said DNA is derived from abacterium belonging to the genus Escherichia.
 10. The method accordingto claim 9, wherein said amino acid is at least one selected from thegroup consisting of L-alanine, L-isoleucine, and L-valine.
 11. Themethod according to claim 9, wherein said DNA is carried on a multicopyvector.
 12. The method according to claim 9, wherein said DNA is carriedon a transposon.
 13. A method for producing an amino acid selected fromthe group consisting of L-threonine, L-homoserine, L-alanine,L-isoleucine, and L-valine comprising: A) cultivating a bacterium whichhas an ability to produce the amino acid in a medium, and B) recoveringthe amino acid from the medium, wherein said bacterium belongs to thegenus Escherichia, wherein L-homoserine resistance of said bacterium isenhanced by amplifying a copy number of a DNA in said bacterium, whereinsaid DNA is derived from a bacterium belonging to the genus Escherichia,wherein said DNA is able to hybridize under stringent conditions tonucleotides 557 to 1171 of SEQ ID NO: 1, wherein said DNA is not lessthan 70% homologous to nucleotides 557 to 1171 of SEQ ID NO: 1, andwherein said DNA encodes a protein which has an activity of impartingL-homoserine resistance to a bacterium having the protein.
 14. Themethod according to claim 13, wherein said amino acid is at least oneselected from the group consisting of L-alanine, L-isoleucine, andL-valine.
 15. The method according to claim 13, wherein said DNA iscarried on a multicopy vector.
 16. The method according to claim 13,wherein said DNA is carried on a transposon.